PECAM-1 (CD31) homophilic interaction up-regulates alpha6beta1 on transmigrated neutrophils in vivo and plays a functional role in the ability of alpha6 integrins to mediate leukocyte migration through the perivascular basement membrane.

Bottom Line:
An anti-alpha(6) integrins mAb (GoH3) inhibited (78%, P < 0.001) neutrophil migration through interleukin (IL)-1beta-stimulated cremasteric venules, primarily at the level of the PBM, as analyzed by intravital and electron microscopy.In PECAM-1-deficient mice (KO), a reduced level of neutrophil transmigration elicited by IL-1beta (4-h reaction) was observed in both the cremaster muscle (55% inhibition, P < 0.05) and in the peritoneum (57% inhibition, P < 0.01) but GoH3 had no additional inhibitory effect on these responses.Furthermore, mice deficient in either leukocyte or endothelial cell PECAM-1, as developed by bone marrow transplantation, demonstrated a similar level of reduced neutrophil transmigration and expression of alpha(6)beta(1) on transmigrated neutrophils as that detected in KO mice.

ABSTRACTPlatelet-endothelial cell adhesion molecule (PECAM)-1 has been implicated in leukocyte migration through the perivascular basement membrane (PBM) though the mechanisms involved are unclear. The present results demonstrate that the ability of alpha(6) integrins to mediate neutrophil migration through the PBM is PECAM-1 dependent, a response associated with PECAM-1-mediated increased expression of alpha(6)beta(1) on transmigrating neutrophils in vivo. An anti-alpha(6) integrins mAb (GoH3) inhibited (78%, P < 0.001) neutrophil migration through interleukin (IL)-1beta-stimulated cremasteric venules, primarily at the level of the PBM, as analyzed by intravital and electron microscopy. In PECAM-1-deficient mice (KO), a reduced level of neutrophil transmigration elicited by IL-1beta (4-h reaction) was observed in both the cremaster muscle (55% inhibition, P < 0.05) and in the peritoneum (57% inhibition, P < 0.01) but GoH3 had no additional inhibitory effect on these responses. FACS((R)) analysis of neutrophils demonstrated increased expression of alpha(6)beta(1) on transmigrated peritoneal neutrophils, as compared with blood neutrophils, in wild-type but not KO mice even though neutrophils from both strains of mice exhibited comparable levels of intracellular expression of alpha(6) as observed by immunofluorescent staining and confocal microscopy. Furthermore, mice deficient in either leukocyte or endothelial cell PECAM-1, as developed by bone marrow transplantation, demonstrated a similar level of reduced neutrophil transmigration and expression of alpha(6)beta(1) on transmigrated neutrophils as that detected in KO mice. The results demonstrate a role for PECAM-1 homophilic interaction in neutrophil transmigration and increased expression of alpha(6)beta(1) on the cell surface of transmigrated neutrophils in vivo, a response that could contribute to the mechanism of PECAM-1-mediated neutrophil migration through the PBM.

fig3: Effect of GoH3 on (A) leukocyte transmigration through IL-1β–stimulated cremasteric venules and (B) neutrophil infiltration into IL-1β–stimulated peritoneal cavity of WT and PECAM-1–deficient mice. Wild-type or PECAM-1–deficient mice were injected with intra-scrotal (A) or intraperitoneal (B) saline (white bars) or intrascrotal or intraperitoneal IL-1β (30 ng/mouse and 10 ng/cavity, respectively), 4 h before quantification. In the IL-1β–injected groups, the mice were pretreated with an isotype-matched control antibody or GoH3 (both at 3 mg/kg intravenously) 15 min before administration of IL-1β. The data represent mean ± SEM from n = 3–6 mice/group in A and n = 5–10 in B. Significant differences between control IL-1β elicited responses in WT and PECAM-1–deficient mice are indicated by +P < 0.05 and ++P < 0.01. Additional statistical comparisons are indicated by lines.

Mentions:
We next examined the effect of mAb GoH3 on neutrophil transmigration in both WT and PECAM-1–deficient mice, using two IL-1β–driven models, namely neutrophil migration through IL-1β–stimulated cremasteric venules and neutrophil migration into IL-1β–stimulated peritoneal cavities. In WT mice, locally administered IL-1β (intrascrotal 30 ng or intraperitoneal 10 ng, administered 4 h before quantification) elicited significant neutrophil transmigration as compared with animals injected with saline (Fig. 3). These responses were almost totally inhibited in mice treated with whole (Fig. 3) or F(ab′)2 fragment of GoH3 (unpublished data). Interestingly, in PECAM-1–deficient mice, while there was a significant suppression of IL-1β–induced neutrophil transmigration in both models (55 and 57% inhibition of neutrophil transmigration in the cremaster muscle and peritoneum, respectively), GoH3 had no additional inhibitory effects (Fig. 3). With respect to the peritonitis model, we have used total and differential leukocyte counts to express the neutrophil migration data as percentage of neutrophil infiltration due to occasional large variations in total leukocyte numbers. However, as the observed suppressed neutrophil migration into IL-1β–stimulated peritoneal cavity of PECAM-1–deficient mice is a novel and important observation, the absolute number of total leukocyte counts, as well as the percent differentials of the infiltrating leukocytes, obtained in response to intraperitoneal PBS or IL-1β, as compared with thioglycollate, in WT and PECAM-1 KO mice, is shown in Table I . The results demonstrate an increase in total infiltrating leukocytes and percentage of neutrophils in response to both IL-1β and thioglycollate in WT mice. In PECAM-1–deficient mice, no significant difference in responses elicited by thioglycollate was observed, as compared with WT mice, in agreement with a previously published report (8). In contrast, a significant suppression of infiltrating neutrophils was detected after intraperitoneal injection of IL-1β, in agreement with the data presented in Fig. 3. Interestingly, the suppression of neutrophil infiltration appeared to be compensated by a significant increase in infiltrating mononuclear leukocytes.

PECAM-1 (CD31) homophilic interaction up-regulates alpha6beta1 on transmigrated neutrophils in vivo and plays a functional role in the ability of alpha6 integrins to mediate leukocyte migration through the perivascular basement membrane.

fig3: Effect of GoH3 on (A) leukocyte transmigration through IL-1β–stimulated cremasteric venules and (B) neutrophil infiltration into IL-1β–stimulated peritoneal cavity of WT and PECAM-1–deficient mice. Wild-type or PECAM-1–deficient mice were injected with intra-scrotal (A) or intraperitoneal (B) saline (white bars) or intrascrotal or intraperitoneal IL-1β (30 ng/mouse and 10 ng/cavity, respectively), 4 h before quantification. In the IL-1β–injected groups, the mice were pretreated with an isotype-matched control antibody or GoH3 (both at 3 mg/kg intravenously) 15 min before administration of IL-1β. The data represent mean ± SEM from n = 3–6 mice/group in A and n = 5–10 in B. Significant differences between control IL-1β elicited responses in WT and PECAM-1–deficient mice are indicated by +P < 0.05 and ++P < 0.01. Additional statistical comparisons are indicated by lines.

Mentions:
We next examined the effect of mAb GoH3 on neutrophil transmigration in both WT and PECAM-1–deficient mice, using two IL-1β–driven models, namely neutrophil migration through IL-1β–stimulated cremasteric venules and neutrophil migration into IL-1β–stimulated peritoneal cavities. In WT mice, locally administered IL-1β (intrascrotal 30 ng or intraperitoneal 10 ng, administered 4 h before quantification) elicited significant neutrophil transmigration as compared with animals injected with saline (Fig. 3). These responses were almost totally inhibited in mice treated with whole (Fig. 3) or F(ab′)2 fragment of GoH3 (unpublished data). Interestingly, in PECAM-1–deficient mice, while there was a significant suppression of IL-1β–induced neutrophil transmigration in both models (55 and 57% inhibition of neutrophil transmigration in the cremaster muscle and peritoneum, respectively), GoH3 had no additional inhibitory effects (Fig. 3). With respect to the peritonitis model, we have used total and differential leukocyte counts to express the neutrophil migration data as percentage of neutrophil infiltration due to occasional large variations in total leukocyte numbers. However, as the observed suppressed neutrophil migration into IL-1β–stimulated peritoneal cavity of PECAM-1–deficient mice is a novel and important observation, the absolute number of total leukocyte counts, as well as the percent differentials of the infiltrating leukocytes, obtained in response to intraperitoneal PBS or IL-1β, as compared with thioglycollate, in WT and PECAM-1 KO mice, is shown in Table I . The results demonstrate an increase in total infiltrating leukocytes and percentage of neutrophils in response to both IL-1β and thioglycollate in WT mice. In PECAM-1–deficient mice, no significant difference in responses elicited by thioglycollate was observed, as compared with WT mice, in agreement with a previously published report (8). In contrast, a significant suppression of infiltrating neutrophils was detected after intraperitoneal injection of IL-1β, in agreement with the data presented in Fig. 3. Interestingly, the suppression of neutrophil infiltration appeared to be compensated by a significant increase in infiltrating mononuclear leukocytes.

Bottom Line:
An anti-alpha(6) integrins mAb (GoH3) inhibited (78%, P < 0.001) neutrophil migration through interleukin (IL)-1beta-stimulated cremasteric venules, primarily at the level of the PBM, as analyzed by intravital and electron microscopy.In PECAM-1-deficient mice (KO), a reduced level of neutrophil transmigration elicited by IL-1beta (4-h reaction) was observed in both the cremaster muscle (55% inhibition, P < 0.05) and in the peritoneum (57% inhibition, P < 0.01) but GoH3 had no additional inhibitory effect on these responses.Furthermore, mice deficient in either leukocyte or endothelial cell PECAM-1, as developed by bone marrow transplantation, demonstrated a similar level of reduced neutrophil transmigration and expression of alpha(6)beta(1) on transmigrated neutrophils as that detected in KO mice.

ABSTRACTPlatelet-endothelial cell adhesion molecule (PECAM)-1 has been implicated in leukocyte migration through the perivascular basement membrane (PBM) though the mechanisms involved are unclear. The present results demonstrate that the ability of alpha(6) integrins to mediate neutrophil migration through the PBM is PECAM-1 dependent, a response associated with PECAM-1-mediated increased expression of alpha(6)beta(1) on transmigrating neutrophils in vivo. An anti-alpha(6) integrins mAb (GoH3) inhibited (78%, P < 0.001) neutrophil migration through interleukin (IL)-1beta-stimulated cremasteric venules, primarily at the level of the PBM, as analyzed by intravital and electron microscopy. In PECAM-1-deficient mice (KO), a reduced level of neutrophil transmigration elicited by IL-1beta (4-h reaction) was observed in both the cremaster muscle (55% inhibition, P < 0.05) and in the peritoneum (57% inhibition, P < 0.01) but GoH3 had no additional inhibitory effect on these responses. FACS((R)) analysis of neutrophils demonstrated increased expression of alpha(6)beta(1) on transmigrated peritoneal neutrophils, as compared with blood neutrophils, in wild-type but not KO mice even though neutrophils from both strains of mice exhibited comparable levels of intracellular expression of alpha(6) as observed by immunofluorescent staining and confocal microscopy. Furthermore, mice deficient in either leukocyte or endothelial cell PECAM-1, as developed by bone marrow transplantation, demonstrated a similar level of reduced neutrophil transmigration and expression of alpha(6)beta(1) on transmigrated neutrophils as that detected in KO mice. The results demonstrate a role for PECAM-1 homophilic interaction in neutrophil transmigration and increased expression of alpha(6)beta(1) on the cell surface of transmigrated neutrophils in vivo, a response that could contribute to the mechanism of PECAM-1-mediated neutrophil migration through the PBM.